1. Field
The present invention relates to the detection and differentiation of small RNAs, and more specifically to methods and compositions for unambiguously delineating mature versus the precursor and primary microRNA in small RNA maturation pathway.
2. Description of the Related Art
Classes of small RNAs include microRNAs (miRs), moRs (miR-offset RNAs), short interfering RNAs (siRNAs), trans-acting siRNAs (tsai RNAs) Piwi interacting RNAs (piRNAs), tRNA derived RNA fragments (tRFs) (Farazi, T A et al., Development 2008, 135, 1201-12140; Lee, Y S. et al., Genes Dev., 2009, 23, 2639-2649) and cleaved miRNA targets, the degradome of mRNA (Addo-Quaye, C. et al., Current Biol., 2008, 18(1), 130-131). In recent years the mature small RNAs, termed the microRNAs (miRNAs) have emerged as important biological regulatory molecules playing a pivotal role in gene expression of plants and animals, including humans. In microRNA maturation pathway, the mature ˜16-22 nucleotides (nts) small RNAs are processed from the poly A tailed primary transcripts of non-coding or introns of coding genes (Faller, M. et al., BBA, 2008, 1779, 663-667; Winter, J. et al., Nature Cell Biol., 2009, 11(3), 228-234). The primary miRNA (pri-miR) is first processed into an intermediate precursor miRNA (pre-miR) and finally into the small mature miR (mat-miR). A typical pri-miR comprises a stem-loop hairpin like secondary structure with ˜33 nt double stranded (ds) stem with long single strand (ss) sequences flanking the base of the stem on one end and a terminal loop at the other end. Both the terminal loop and the single strand flanking regions have been found to be important in the processing of pri-miR to the precursor intermediates (Zeng, Y. et al., Jour. Biol. Chem., 2005, 280(30), 27595-27603; Han, J. et al., Cell 2006, 125, 887-901).
The pri-miR is processed into a stem-loop precursor miRNA (pre-miR) in the nucleus by Drosha/RNASEN, a ribonuclease III (RNase III) and DGCR8/Pasha, a double stranded RNA binding cofactor protein (Han, J. et al., Genes & Deve., 2004, 18, 3016-3027). The Drosha-DGCR8 complex, collectively termed the microprocessor recognizes the structural features of the pri-miRNA. DGCR8 positions Drosha to cleave pri-miR ˜11 bp within the stem region measuring from the ds stem-ssRNA junction yielding the pre-miR intermediate. Drosha cleavage of the pri-miR takes place co-transcriptionally within the nucleus (Morlando, M. et al., Nature Struc. & Mol. Biol., 2008, 15(9), 902-909). Recent evidence suggests that Drosha cleavage of the pri-miR in the pre-miR-proximal regions also results in another distinct class of small RNAs termed the moRs (miR-offset RNAs) (Shi, W. et al., Nature struc. Mol. Biol., 2009, 16(2), 183-189). The pre-miRNA is also generated from spliced introns of mRNA (Winter, J. et al., Nat Cell Biol., 2009, 11(3), 228-234). The pre-miRNA is then transported and further processed in the cytosol by another RNase III enzyme, the Dicer which removes the terminal loop region to yield the small ˜22 nt double stranded mature miRNA. One of the strands of the duplex miRNA is then selectively retained within another enzyme, the RISC complex, resulting in the functional mature miRNA (mat-miR). The processing and expression of micro RNAs can be further modulated by RNA editing enzymes such as the ADAR deaminases (Yang, W., et al., Nature Struc. & Mol. Biol., 2006, 13(1), 13-21). The mature miRNAs regulate mRNA expression at the translational level by binding to the target mRNA at their 3′ UTR region, thus regulating gene expression profiles. In this way, miRNAs have been implicated as global modulators of diverse cellular and biological functions from proliferation to development, differentiation and apoptosis, and therefore seem to play a significant role in oncogenesis.
Expression profiles of a few hundred miRNA have provided more specific classification of human cancers than mRNA profiles (Lu, J., et al., Nature, 2005, 435(7043), 834-838). Unique expression profiles identified in lung cancers position miRNA as diagnostic and prognostic markers (Yanaihara, N., et al., Cancer Cell, 2006, 9(3), 189-198). Decreased levels of the microRNAs miR-143 and miR-145 are displayed consistently in colorectal neoplasia as well as in lung and breast cancers. In chronic lymphocytic leukemia (CLL) levels of miR-15a and miR-16-1 are decreased, and these rare germ-line mutations are thought to be prognostic. The miRNA let-7 family members that are negative regulators of Ras oncogene have been found to be decreased in lung cancers (Takamizawa, J., et al., Cancer Res., 2004, 64(11), 3753-3756). Impairment in miRNA processing pathway due to reduced expression of Dicer has been implicated in lung cancers. Furthermore, it was reported that failure of Drosha processing in primary tumors accounts for global down regulation of mature miRNAs (Chng, W. J., N Engl J Med, 2006, 354(5), 524-525). Profiles of miRNA in cancer cells is analogous to embryonic stem cells and perhaps play a role in maintaining a gentle balance between development and disease (Hammond, S. M., Nat Methods, 2006, 3(1), 12-13; Lao, K., et al., Biochem. Biophys. Res. Com., 2006, 343(1), 85-89). Importantly, five members of miR-200 family and miR-205 are markedly down regulated in cells that have undergone epithelial to mesenchymal transition (EMT) and are lost in invasive breast cancer cell lines with mesenchymal phenotype (Gregory, P. A. et al., Nat. Cell Biol. 2008, 10(5), 593-601); and up-regulation of miR-10b and down-regulation of miR-335, miR-126 and miR-206 are shown to promote tumor metastasis (Brackena, C. P. et al., 2009 Cell. Mol. Life Sci. DOI 10.1007/s00018-009-8750-1). Thus miRNAs as molecular markers reflect a cell's altered biological state.
The current status signifying an important role for miRNA in the disease and normal state is replete with such examples. Nevertheless, detecting miRNAs have posed several challenges, not only due to their extremely low levels of expression (0.001%) within cells; but, also owing to their dynamic range, as well as extremely small size making the balancing of the melting temperatures (Tm) in the design of primers for detection quite challenging. Importantly the high sequence homology in families of miRNA, isomiRs which are variant length forms at the 3′ and 5′ end of mature miRNA (Morin R D et al. Genome Res. 2008, 18:610-621; Kuchenbauer et al., Genome Res., 2008, 18:1787-1797) and also segments of sequence identity in the pri-miRNA, pre-miRNA and the mat-miRNA of the microRNA maturation pathway, further compound the challenges. From a molecular diagnostic perspective specificity in detecting mature miRNA versus the precursor miRNA and also the precursor versus the primary miRNA species of the small RNA maturation pathway is a useful determinant in defining the state and stage of cancer and other diseases.
Several strategies that are applicable for detecting the long mRNAs have not proven directly suitable for use in detecting and characterizing small RNAs, such as the miRNA. As mentioned, a major limitation is designing appropriate and specific primers to capture the different miRNA species; some aspects of which are addressed using LNA based primers (Lunn, M. L. et al., Nature Methods 2008, 5, doi: 10.1038/nmeth.f.205). The specificity in unambiguous detection of mature miRNA products in several design strategies and methods described in the prior art including microarrays (Agilent, Ambion, Genispere, Invitrogen), bead-based assays (Luminex), qRT-PCR (ABI), TMA assay (U.S. Pat. No. 7,374,885) and NEAR assay (US 20090017453) is questionable; because, the methods in the prior art seem to address only detection of mature miRNA versus the primary miRNA sequence structure of the type annotated in the Sanger Database miRBase Registry (Griffith-Jones, S. et al., Nucleic Acids Res., 2006, 34 (Database Issue), D140-D144); and not mature miRNA versus the actual intermediate precursor miRNA formed in accordance with the biogenesis of miRNA maturation pathway via the routes of both Drosha-DGCR8 and spliceosome processing. Furthermore, the precise sequence of the precursor i.e. the pre-miRs which is the substrate for Dicer is not annotated in miRBase Registry (miRBase version 14); and due to similarity of the pre-miR sequence to that of the pri-miR sequence, pre-miR detection is often grouped together with pri-miR and levels of pre-miRs expression is quantified indirectly (Schmittgen, T. D. et al., Nucleic Acids Res, 2004, 32(4), e43; US 20090123917). The ambiguity in detecting mature miRNA is further compounded due to the technical variability in high-throughput miRNA expression profiling methods (Nelson, P. T. et al., Biochem. Biophy. Acta, 2008, 1779, 758-765). Cloning and sequencing, non-Sanger based ‘sequencing while synthesis’ next generation sequencing (NGS) platforms and ‘Deep Sequencing’ technologies (Solexa/Illumina and 454 Life Sciences/Roche) which offer unprecedented throughputs for miRNA profiling with high resolution views (Chen J. et al., Nucleic Acids Res., 2008, 36(14), e87; Friedländer, M. R. et al., Nature Biotech., 2008, 26(4), 407-415; Blow N., Nature Methods, 2009, 6(3), 231-234) would perhaps help resolve this ambiguity in detection of the small RNAs. Such sequencing technology is however ultrahigh in the complexity of the information generated and processing for clinical diagnostic settings.
The quantitative RT-PCR (qRT-PCR) method, though known as the ‘gold standard’ for mRNA detection/characterization (Bustin, S. A., J Mol Endocrinol, 2000, 25(2), 169-193), has not been directly translatable for detecting miRNA. One of the modifications adapted in qRT-PCR for detecting miRNA is the stem-loop reverse primer with 6 nt single strand primer binding region that complements and binds to the 3′ end of the target miRNAs (Chen, C., et al., Nucleic Acids Res, 2005, 33(20), e179) for reverse transcription and subsequent amplification by PCR. One important consideration in this scheme is that the stem-loop reverse primer (RP) design would prevent binding of precursor/primary miRNA due to the bulky secondary loop structure at the 3′ end. It is assumed that the stearic hindrance would preclude binding of the reverse primers to the precursor miRNA and hence aid specifically in the detection of all mature miRNAs. The reverse primer stem-loop scheme possibly would be selective against pre-miRNAs and preferentially aid in detecting mature miRNAs only if mature miRNA results from the 5′ end of the precursor miRNA stem and not if the mature miRNA results from its 3′ end. In miRNA maturation pathway mature miRNAs are processed from both the 5′ end (miR-5p) as well as the 3′ end (miR-3p) of the precursor pre-miRNA. Furthermore, the sequence structure of the intermediate precursor miRNA (Zeng et al., JBC, 2005, 280(30), 27595-27603; Yang et al., Nature Struc. & Mol. Biol., 2006, 13(1), 13-21) suggests that the reverse primer used for detecting miR-3p would anneal to the 3′ end of both the mature miRNA and the intermediate precursor pre-miR, thus producing cDNA and signal from both targets in precisely the same manner; which is henceforth termed as the “3′ bias” in mature miR-3p detection in this invention Furthermore, isomiRs which are 3′ and 5′ length variant forms would add further ambiguity to detection by taqman RT_PCR (Morin R D et al. Genome Res. 2008, 18:610-621). Accordingly, methods based on RT-PCR schemes either with stem-loop reverse primer (Lao, K., et al., Biochem Biophys Res Commun, 2006, 343(1), 85-89; US 20050266418) or linear reverse primer (Raymond, C. K., et al., RNA, 2005, 11(11), 1737-1744; US 20080131878) may not differentiate all of the mature miR products processed from pre-miR precursor unambiguously.
The suggested application of NEAR assay (US 20090081670) for small RNA detection would also result in non-specific detection of the different mature miRNA species; as the method would permit amplification of the mature miRNA sequence embedded within the precursor and primary miRNA species as an amplicon, thus losing specificity and biasing the qualitative and quantitative profiling of mature miRNA. Similar problems would be encountered in other methods described for miRNA detection (US 20080051296).